The invention relates to compositions and methods for generating and using pIX phage display libraries for producing antibodies or antibody fragments.
Filamentous phage display using pIII and pVIII as fusion partners in phage or phagemid systems have been used as a technology for protein engineering, notably for de novo antibody isolation and affinity maturation. Human-like Fab sequences can be generated from known antibody template sequences and random or mutangenized complementarity determining regions (CDR) or antigen binding regions, such as heavy chain CDR3 (H3). Such sequences can be generated and isolated from phage libraries displaying variations of antibody fragment sequences via panning against an antigen or other target of interest without immunization. Previously used human de novo antibody libraries have been created synthetically or by molecular cloning of IgG genes from naive source(s) or by combinations of both methods. In a synthetic library, antibody DNA sequences including variable heavy chain and light chain framework sequences, and the CDR regions, are designed and synthesized based on 1) a defined IgG gene; 2) a specific Ig germline gene; 3) consensus sequences from families of the Ig germline genes; and/or 4) PCR-derived IgG fragments from natural sources. Libraries may also be created by combinatorial cloning of IgG DNA derived from human tissues, e.g. bone marrow and peripheral blood cells. Such libraries have been used for providing Fab antibody fragments (i.e., the antigen-binding fragment of an immunoglobulin molecule, comprising both a light chain and part of a heavy chain) and for running successive rounds of panning and maturation or modification to find Fab antibody fragments that have desired properties such as high affinity target binding or the ability to modulate the biological activity of a selected target protein.
Human-like Fabs that bind target proteins have been isolated from phage display pIII or pVIII de novo libraries. Although successful at isolating Fabs that bind to specific targets, such phage display library approaches often suffer from the problem of having to repeat the process of library generation, panning for Fabs that bind to a target of interest, and maturing the Fabs one or more times to isolate Fab antibodies having the desired characteristics. Some phage libraries also suffer from the problem that they do not fully encompass or mimic the full range of human immune diversity, including the contribution of VH/VL paring, topology features of different germline families related to antigen recognition, the position and extent of amino acid variation, and the relative abundance of antibodies derived from different human germline genes. Deviation of synthetic antibodies from the natural repertoire may increase the risk of unfavorable biochemical properties and of immunogenicity if used as therapeutics in humans.
Counter to prior conclusions (See e.g., Endemann and Model, J. Mol. Biol., 250: 496-506, 1995), the filamentous phage proteins pVII and pIX were found to localize on the phage surface in an orientation suitable for protein and peptide display (Gao et al., Proc Natl Acad Sci USA, 96: 6025-6030, 1999). Subsequently, the pIX protein was utilized for display of a single chain antibody library (Gao et al., Proc Natl Acad Sci USA, 99: 12612-12616, 2002). However, this approach was not successful for construction and display of Fab libraries.
There is a need for synthetic antibody libraries and methods that simultaneously deliver the elements of human therapeutic antibodies of high affinity and activity, high productivity, good solution properties, and a propensity of low immune response when administered in humans. There is a further need to increase the efficiency of antibody isolation from synthetic libraries, relative to current methods, to reduce the resource costs of antibody discovery and accelerate delivery of antibodies for biological evaluation. The libraries and methods of this invention meet these needs by coupling comprehensive design, assembly technologies, and phage pIX Fab display.